One of the main problems with designing a gene therapy approach for a continuously renewing tissue, such as the epidermis of the skin, is that most of the cells transfected or transduced with the target genes are eventually sloughed into the environment (Morgan, 1987; Vogt, 1994; Huber, 1995; Choate, 1996; Fenjives, 1996; Freiberg, 1997; Dellambra, 1998; Seitz, 1999). Epidermal tissue integrity is maintained by division of cells in the proliferative basal layer to replace differentiated cells in the outermost stratum corneum layer that are continually lost. In human and mouse epidermis, most of the cells are replaced every twenty days (Gelfant, 1982). This hierarchy of cell proliferation and differentiation is maintained by a small subpopulation of stem cells (for a review see Potten, 1997), which slowly proliferate. It has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label. It has been assumed that epidermal stem cells are a self-renewing population in which, on average, each cell division produces one stem cell and one transient amplifying (TA) cell. The TA cells, as their name implies, amplify the basal cell population, but they are limited to a finite number of cell divisions before their progeny must commit to differentiate and are ultimately sloughed into the environment. Only the stem cells remain and continue to proliferate for the lifetime of the epidermis (Cairns, 1975). This implies that any genetic treatment must be directed toward the stem cell genome. Unfortunately, isolating a pure population of epidermal stem cells has been problematic.
The main function of the skin is to provide a protective barrier for the internal tissues of the body. When this barrier is broken, it can be life-threatening as the body loses fluids and is exposed to harmful factors in the environment. Allogeneic grafts made from cultured epidermal cells that had been expanded at least ten-fold have been shown to last for years (Rheinwald, 1975; Otto, 1993; Compton, 1989. Although these grafts do not usually form all of the epidermal appendages, such as hair follicles and sweat glands, they reform a structured epidermis with spinous, granular, and cornified cells overlying the basal cell compartment, suggesting that epidermal stem cells survived and replicated during the culturing procedure.
The next steps are to use epidermal stem cells to bioengineer a complete skin and to use epidermal stem cells to deliver genes for permanent gene therapy. For delivery of genes to a variety of cell types, replication deficient viral vectors have been the major vehicles (Garlick, 1991; Vogt, 1994; Ng, 1997). Using a retroviral-LacZ gene for transduction into the epidermis, several investigators reported good initial expression in keratinocytes, but disappearance within 14 to 40 days (Morgan, 1987; Vogt, 1994; Fenjives, 1996). Loss of expression was thought to be due to selective methylation of viral promoters (Fenjives, 1996) or to the loss of the transduced cells (Kolodka, 1998). In support of the latter view, recent studies have reported migration of transduced cells through the differentiated layers of the epidermis, with only a few keratinocytes showing long term expression (Flowers, 1990; Mackenzie, 1997; Deng, 1997; Ghazizadeh, 1999). Thus, it seems likely that total loss of expression in a tissue may be due to the failure to transduce stem cells. This might happen because stem cells make up a very small percentage of the basal cell population. Thus, any method that increases the percentage of stem cells in the population should increase the possibility of transfecting stem cells, thereby improving gene therapy approaches.
Thus, what is needed is a method to prepare a population of substantially pure epidermal stem cells, which cells are useful to form tissues or to deliver genes.